The RAS gene is centrally important to the development of melanoma. It is mutated in only 10% of cases, but cooperates with mutations in CDKN2A in both human and murine melanomas. RAS proteins mediate their effects biochemically through several downstream pathways, but most importantly through the mitogenactivated protein kinase (MAPK) cascade, or through the control of protein kinase B/Akt via phosphoinositol-3-kinase (PI3K). Recently it has been demonstrated that in melanoma both of these pathways are affected by a high incidence of mutation. Mutations on the Akt pathway involve PTEN. PTEN is a tumor suppressor that shares several characteristics with RAS. It is a protein tyrosine phosphatase, but also has lipid phosphatase activity. Thus it is a negative regulator of PI3K and an effector of apoptosis through PKB/Akt. We have demonstrated that PTEN loss in melanoma is a frequent event, occurring in about 30% of specimens examined. And recently, the parallel pathway was also shown to be involved by mutation. BRAF lies immediately downstream of RAS on the MAPK cascade. Mutations in BRAF have been recently found in over 60% of melanomas. Data presented here demonstrate that most of these BRAF mutations occur with PTEN abnormalities or other Akt-pathway aberrations. In turn, mutations in BRAF/PTEN occur 1) reciprocally with RAS mutations, and 2) like RAS, in concert with CDKN2A loss. This suggests the hypothesis that the salient biochemical functions of RAS in melanoma development are encompassed by mutations in these two cancer genes. We propose four specific aims to test this. First, we will model our genetic observations in the mouse, and predict Braf activation and Pten loss with Cdkn2a deficiency in the mouse will engender the development of melanoma. We will generate mice carrying Pten and Braf alterations in the background of p16 and p53 deficiency. Second, we will examine RAS or BRAF and PTEN alterations separately and together in the transgenic murine lines, melanocytes, melanoma and other cell types using genetic and pharmacologic strategies. Third, since alterations in these genes occur in the background of abrogation of p16 function, we predict PTEN and BRAF, like RAS, will exert control over p16 expression, and will assess the control of p16 by these proteins using murine knockout lines. And fourth, we will develop genetic techniques using RNA interference to test strategies for melanoma inhibition and pharmacological therapy.